Transducin beta-1 subunit

ABSTRACT

The invention provides a human transducin beta-1 subunit (TBS) and polynucleotides which identify and encode TBS. The invention also provides expression vectors, host cells, agonists, antibodies, and antagonists. The invention also provides methods for treating or preventing diseases associated with expression of TBS.

This application is a divisional application of U.S. application Ser.No. 08/965,600, filed Nov. 6, 1997.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of atransducin beta-1 subunit and to the use of these sequences in thediagnosis, prevention, and treatment of diseases associated with cellproliferation.

BACKGROUND OF THE INVENTION

Membrane-bound GTP-binding (G) proteins mediate signal transduction in avariety of cell systems. Although, the exact mechanisms of G proteinaction are still under investigation, G proteins appear to involveeffectors located in the plasma membrane as well as other parts of thecell. WD-40 proteins contain a loosely conserved repeat of approximately40 amino acids separated by a Trp-Asp dipeptide sequence which may recurseveral times within each polypeptide. The conserved core of the WD-40sequence, which usually ends with the amino acids Trp-Asp (WD), wasfirst identified in the β-subunit of the heterotrimeric G protein,transducin. Several dozen WD-40 proteins have since been identified,none are enzymes, and all seem to have regulatory functions (Neer, E. J.et al. (1994) Nature 371:297-300).

Many of the WD-40 proteins are homologs of β-transducin and function insignal transduction pathways within the cytoplasm. WD-40 proteins mayparticipate in complex formation, and may interact with other G proteinsubunits through the WD-40 region. The β subunit of G proteins enhancesbinding of the G_(α) subunit to receptors and assists in the assembly ofthe G-protein:receptor complex. The G_(βγ) subunit binds to and bringsthe β-adrenergic receptor kinase, β-ARK, to the receptor (Touhara, K. etal. (1994) J. Biol. Chem. 269: 10217-10220).

A number of WD repeat proteins are localized to the nucleus and functionin the repression of transcription. These include Tup1, Hir1, and Met30in S. cerevisiae; SCON2 in Neurospora crassa; extra sex combs andGroucho in Drosophila; COP1 in Arabidopsis thaliana; and Rec14 inSchizosaccharomyces pombe. These WD-40 proteins turn off a wide varietyof genes, including those involved in segmentation, sex determination,and neurogenesis, and those involved in photomorphogenesis.

The WD repeat protein, Rec14, is essential for meiotic recombination inS. pombe. Mutations in the Rec14 gene reduce meiotic recombination by asmuch as a factor of 1000 on chromosomes I and III. The Rec14 genecontains two exons separated by a 53-bp intron. The spliced transcriptencodes a protein of 302 amino acids and contains six WD repeat motifscommon in the G-beta transduction family and in S. cerevisiae Rec103protein. Rec103 mutations have no detectable effect on mitoticrecombination. Based upon phenotypes and amino acid sequencesimilarities, it is possible that R is a functional homolog of S.cervisiae Rec103 (Evens, D. H. et al. (1997) Genetics 146:1253-1264).Autoantibodies occurring spontaneously in certain cancer patients arereagents for identifying cellular proteins, and it is the WD-40 motif inthe beta-transducin proteins which target these antibodies.

The discovery of a new transducin beta-1 subunit and the polynucleotidesencoding it satisfies a need in the art by providing new compositionswhich are useful in the diagnosis, prevention and treatment of diseasesassociated with cell proliferation, in particular, cancers and immuneresponse.

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide, transducinbeta-1 subunit (TBS), having the amino acid sequence shown in SEQ IDNO:1, or fragments thereof.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or fragments thereof and a compositioncomprising said polynucleotide sequence. The invention also provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence encoding the amino acid sequence SEQ IDNO:1, or fragments of said polynucleotide sequence. The inventionfurther provides a polynucleotide sequence comprising the complement ofthe polynucleotide sequence encoding the amino acid sequence of SEQ IDNO:1, or fragments or variants of said polynucleotide sequence.

The invention also provides an isolated and purified sequence comprisingSEQ ID NO.2 or variants thereof. In addition, the invention provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of SEQ ID NO:2. The invention also providesa polynucleotide sequence comprising the complement of SEQ ID NO:2, orfragments or variants thereof.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding TBS under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified TBS having the amino acid sequence of SEQ ID NO:1in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist of the polypeptide ofSEQ ID NO:1. In one aspect the invention provides a purified antibodywhich binds to a polypeptide comprising the amino acid sequence of SEQID NO:1.

Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:1.

The invention also provides a method for treating or preventing animmune response disorder comprising administering to a subject in needof such treatment an effective amount of an antagonist to TBS.

The invention also provides a method for treating or preventing a cancercomprising administering to a subject in need of such treatment aneffective amount of an antagonist to TBS.

The invention also provides a method for detecting a polynucleotidewhich encodes TBS in a biological sample comprising the steps of: a)hybridizing the complement of the polynucleotide sequence which encodesSEQ ID NO:1 to nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting the hybridizationcomplex, wherein the presence of the complex correlates with thepresence of a polynucleotide encoding TBS in the biological sample. Inone aspect the nucleic acid material of the biological sample isamplified by the polymerase chain reaction prior to hybridization.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C and 1D show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of transducin beta-1 subunit (TBS).The alignment was produced using MacDNASIS PRO™ software (HitachiSoftware Engineering Co. Ltd. San Bruno, Calif.).

FIG. 2 shows the amino acid sequence alignments among TBS (194046; SEQID NO:1) and Rec14 (GI 1079671; SEQ ID NO:3), produced using themultisequence alignment program of DNASTAR™ software (DNASTAR Inc,Madison Wis.).

FIGS. 3A and 3B show the hydrophobicity plots for TBS (SEQ ID NO:1) andRec14 (SEQ ID NO:3), respectively; the positive X axis reflects aminoacid position, and the negative Y axis, hydrophobicity (MacDNASIS PROsoftware).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

TBS, as used herein, refers to the amino acid sequences of substantiallypurified TBS obtained from any species, particularly mammalian,including bovine, ovine, porcine, murine, equine, and preferably human,from any source whether natural, synthetic, semi-synthetic, orrecombinant.

The term “agonist”, as used herein, refers to a molecule which, whenbound to TBS, increases or prolongs the duration of the effect of TBS.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of TBS.

An “allele” or “allelic sequence”, as used herein, is an alternativeform of the gene encoding TBS. Alleles may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding TBS as used herein includethose with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent TBS. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding TBS, andimproper or unexpected hybridization to alleles, with a locus other thanthe normal chromosomal locus for the polynucleotide sequence encodingTBS. The encoded protein may also be “altered” and contain deletions,insertions, or substitutions of amino acid residues which produce asilent change and result in a functionally equivalent TBS. Deliberateamino acid substitutions may he made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the biological orimmunological activity of TBS is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine,glycine and alanine, asparagine and glutamine, serine and threonine, andphenylalanine and tyrosine.

“Amino acid sequence” as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragment thereof, and to naturallyoccurring or synthetic molecules. Fragments of TBS are preferably about5 to about 15 amino acids in length and retain the biological activityor the immunological activity of TBS. Where “amino acid sequence” isrecited herein to refer to an amino acid sequence of a naturallyoccurring protein molecule, amino acid sequence, and like terms, are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

“Amplification” as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term “antagonist” as used herein, refers to a molecule which, whenbound to TBS, decreases the amount or the duration of the effect of thebiological or immunological activity of TBS. Antagonists may includeproteins, nucleic acids, carbohydrates, antibodies or any othermolecules which decrease the effect of TBS.

As used herein, the term “antibody” refers to intact molecules as wellas fragments thereof, such as Fa, F(ab′)₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind TBS polypeptidescan be prepared using intact polypeptides or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide oroligopeptide used to immunize an animal can be derived from thetranslation of RNA or synthesized chemically and can be conjugated to acarrier protein, if desired. Commonly used carriers that are chemicallycoupled to peptides include bovine serum albumin and thyroglobulin,keyhole limpet hemocyanin. The coupled peptide is then used to immunizethe animal (e.g., a mouse, a rat, or a rabbit).

The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

The term “biologically active”, as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic TBS, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms “complementary” or “complementarity”, as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

A “composition comprising a given polynucleotide sequence” as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding TBS (SEQ ID NO:1) or fragments thereof (e.g., SEQ ID NO:2 andfragments thereof) may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

“Consensus”, as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, has been extended usingXL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′ directionand resequenced, or has been assembled from the overlapping sequences ofmore than one Incyte Clone using a computer program for fragmentassembly (e.g., GELVIEW™ Fragment Assembly system, GCG, Madison, Wis.).Some sequences have been both extended and assembled to produce theconsensus sequence.

The term “correlates with expression of a polynucleotide”, as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 by northern analysis is indicative of thepresence of mRNA encoding TBS in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

A “deletion”, as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides.

The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to TBS or theencoded TBS. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Human artificial chromosomes (HACs) are linear microchromosomes whichmay contain DNA sequences of 10K to 10M in size and contain all of theelements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The term “hybridization”, as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term “hybridization complex”, as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

An “insertion” or “addition”, as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

“Microarray” refers to an array of distinct polynucleotides oroligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

The term “modulate”, as used herein, refers to a change in the activityof TBS. For example, modulation may cause an increase or a decrease inprotein activity, binding characteristics, or any other biological,functional or immunological properties of TBS.

“Nucleic acid sequence” as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. “Fragments” are thosenucleic acid sequences which are greater than 60 nucteotides than inlength, and most preferably includes fragments that are at least 100nucleotides or at least 1000 nucleotides, and at least 10,000nucleotides in length.

The term “oligonucleotide” refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or a hybridization assay, or a microarray.As used herein, oligonucleotide is substantially equivalent to the terms“amplimers”,“primers”, “oligomers”, and “probes”, as commonly defined inthe art.

“Peptide nucleic acid”, PNA as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast five nucleotides in length linked to a peptide backbone of aminoacid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

The term “portion”, as used herein, with regard to a protein (as in “aportion of a given protein”) refers to fragments of that protein. Thefragments may range in size from five amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein “comprising atleast a portion of the amino acid sequence of SEQ ID NO:1” encompassesthe full-length TBS and fragments thereof.

The term “sample”, as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding TBS, orfragments thereof, or TBS itself may comprise a bodily fluid, extractfrom a cell, chromosome, organelle, or membrane isolated from a cell, acell, genomic DNA, RNA, or cDNA(in solution or bound to a solid support,a tissue, a tissue print, and the like.

The terms “specific binding” or “specifically binding”, as used herein,refers to that interaction between a protein or peptide and an agonist,an antibody and an antagonist. The interaction is dependent upon thepresence of a particular structure (i.e., the antigenic determinant orepitope) of the protein recognized by the binding molecule. For example,if an antibody is specific for epitope “A”, the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled “A” and the antibody will reduce the amount of labeled A boundto the antibody.

The terms “stringent conditions” or “stringency”, as used herein, referto the conditions for hybridization as defined by the nucleic acid,salt, and temperature. These conditions are well known in the art andmay be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

The term “substantially purified”, as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

A “substitution”, as used herein, refers to the replacement of one or.more amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

A “variant” of TBS, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have “nonconservative” changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

The Invention

The invention is based on the discovery of a new human transducin beta-1subunit (hereinafter referred to as “TBS”), the polynucleotides encodingTBS, and the use of these compositions for the diagnosis, prevention, ortreatment of cancers and immune response.

Nucleic acids encoding the TBS were first identified in Incyte Clone194046 from the kidney cDNA library (KIDNNOT02) using a computer searchfor amino acid sequence alignments. A consensus sequence, SEQ ID NO:2,was derived from the following overlapping and/or extended nucleic acidsequences: Incyte Clones 194046 (KIDNNOT02), 2550636 (LUNGTUT06), 104214(BMARNOT02), and 1857141 (PROSNOT18).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, and1C. TBS is 305 amino acids in length and has six potential casein kinaseII phosphorylation sites at residues S₃₉, S₈₂, T₂₀₇, S₂₅₀, T₂₆₉, andS₂₉₁; five potential protein kinase C phosphorylation sites at residuesT₂₆, S₁₃₇, T₁₇₉, S₂₅₂, and S₂₅₅; and one potential beta-transductionfamily WD repeats signature site at residue A₇₉. As shown in FIG. 2, TBShas chemical and structural homology with Rec14 (GI 1079671; SEQ IDNO:3). In particular, TBS and Rec14 share 32% identity. As illustratedby FIGS. 3A and 3B, TBS and Rec14 have rather similar hydrophobicityplots. Northern analysis shows the expression of this sequence invarious libraries, at least 58% of which are immortalized or cancerous,at least 20% of which involve call proliferation, and at least 18% ofwhich involve immune response.

The invention also encompasses TBS variants. A preferred TBS variant isone having at least 80% and, more preferably at least 90% amino acidsequence identity to the TBS amino acid sequence (SEQ ID NO:1) andretaining at least one biological, immunological, or other functionalcharacteristic or activity of TBS. A most preferred TBS variant is onehaving at least 95% amino acid sequence identity to SEQ ID NO:1.

The invention also encompasses polynucleotides which encode TBS.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of TBS can be used to produce recombinant molecules whichexpress TBS. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2, asshown in FIGS. 1A, 1B, and 1C.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding TBS, some bearing minimal homology to the nucleotide sequencesof any known and naturally occurring gene, may be produced. Thus, theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequence ofnaturally occurring TBS, and all such variations are to be considered asbeing specifically disclosed.

Although nucleotide sequences which encode TBS and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring TBS under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding TBS or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding TBS and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode TBS and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding TBS or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency as taught in Wahl, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

Methods for DNA sequencing which are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE Amplification Systemmarketed by Gibco/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (PerkinElmer).

The nucleic acid sequences encoding TBS may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devisecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. Genotyper™ and Sequence Navigator™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode TBS may be used in recombinant DNAmolecules to direct expression of TBS, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressTBS.

As will be understood by those of skill in the art, it may beadvantageous to produce TBS-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter TBS encodingsequences for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding TBS may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of TBS activity, it may be useful toencode a chimeric TBS protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the TBS encoding sequence and theheterologous protein sequence, so that TBS may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding TBS may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of TBS, or a fragment thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of TBS, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active TBS, the nucleotide sequencesencoding TBS or functional equivalents, may be inserted into appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding TBS andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding TBS. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene,LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding TBS,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for TBS. For example, when largequantities of TBS are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding TBS may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding TBS may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196.

An insect system may also be used to express TBS. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding TBS may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of TBS will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which TBS may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding TBS may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing TBS in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding TBS. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding TBS, its initiation codon, and upstream sequences are insertedinto the appropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressTBS may be transformed using expression vectors which may contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the arninoglycosidesneomycin and G418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsufiuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding TBS isinserted within a marker gene sequence, transformed cells containingsequences encoding TBS can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding TBS under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding TBS and express TBS may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding TBS can be detected byDNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding TBS. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding TBS to detect transformantscontaining DNA or RNA encoding TBS.

A variety of protocols for detecting and measuring the expression ofTBS, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson TBS is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding TBS includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding TBS, or anyfragments thereof may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio).Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding TBS may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeTBS may be designed to contain signal sequences which direct secretionof TBS through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding TBS to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and TBS may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingTBS and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying TBS from the fusion protein. A discussion of vectors whichcontain fusion proteins is provided in Kroll, D. J. et al. (1993; DNACell Biol. 12:441-453).

In addition to recombinant production, fragments of TBS may be producedby direct peptide synthesis using solid-phase techniques Merrifield J.(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer). Various fragments of TBS may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

Therapeutics

Chemical and structural homology exists between TBS and Rec14 fromSchizosaccharomyces pombe (GI 1079671). Northern analysis shows that theexpression of TBS is associated with cell proliferation, and, inparticular, with the presence of cancers, and immune response disorders.

Therefore, in one embodiment, an antagonist of TBS may be administeredto a subject to treat or prevent an immune response disorders of anytype and, in particular, that which is associated with a particulardisorder. Such disorders include, but are not limited to, AIDS,Addison's disease, adult respiratory distress syndrome, allergies,anemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn'sdisease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjögren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, and extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections; and trauma. In one aspect, an antibody which specificallybinds TBS may be used directly as an antagonist or indirectly as atargeting or delivery mechanism for bringing a pharmaceutical agent tocells or tissues which express TBS.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding TBS may be administered to a subject to treat orprevent an immune response as associated with, but not limited to, thedisorders described above.

In another embodiment, an antagonist of TBS or a fragment or derivativethereof may be administered to a subject to treat or prevent a cancer.Such cancers include, but are not limited to, adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, and, inparticular, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus. In one aspect, an antibody which specifically binds TBS may beused directly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissues whichexpress TBS.

In another embodiment, a vector capable of expressing the complement ofthe polynucleotide encoding TBS may be administered to a subject totreat or prevent a cancer including, but not limited to, those cancerslisted above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of TBS may be produced using methods which are generallyknown in the art. In particular, purified TBS may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind TBS.

Antibodies to TBS may be generated using methods that are well known inthe art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith TBS or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to TBS have an amino acid sequence consisting of atleast five amino acids and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of TBS amino acids may be fused with those of another proteinsuch as keyhole limpet hemocyanin and antibody produced against thechimeric molecule.

Monoclonal antibodies to TBS may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, s but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:3142; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceTBS-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter,G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for TBS may alsobe generated. For example, such fragments include, but are not limitedto, the F(ab′)2 fragments which can be produced by pepsin digestion ofthe antibody molecule and the Fab fragments which can be generated byreducing the disulfide bridges of the F(ab′)2 fragments. Alternatively,Fab expression libraries may be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between TBS and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering TBS epitopes is preferred, but a competitive bindingassay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingTBS, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingTBS may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding TBS. Thus,complementary molecules or fragments may be used to modulate TBSactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments, can be designed from various locations along thecoding or control regions of sequences encoding TBS.

Expression vectors derived from retro viruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encoding TBS.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

Genes encoding TBS can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes TBS. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′ or regulatory regions of the gene encoding TBS(signal sequence, promoters, enhancers, and introns). Oligonucleotidesderived from the transcription initiation site, e.g., between positions−10 and +10 from the start site, are preferred. Similarly, inhibitioncan be achieved using “triple helix” base-pairing methodology. Triplehelix pairing is useful because it causes inhibition of the ability ofthe double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Thecomplementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding TBS.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding TBS. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections orpolycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of TBS, antibodies to TBS,mimetics, agonists, antagonists, or inhibitors of TBS. The compositionsmay be administered alone or in combination with at least one otheragent, such as stabilizing compound, which may be administered in anysterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations.which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of TBS, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example TBS or fragments thereof, antibodies of TBS,agonists, antagonists or inhibitors of TBS, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind TBS may beused for the diagnosis of conditions or diseases characterized byexpression of TBS, or in assays to monitor patients being treated withTBS, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for TBS includemethods which utilize the antibody and a label to detect TBS in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuring TBSare known in the art and provide a basis for diagnosing altered orabnormal levels of TBS expression. Normal or standard values for TBSexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toTBS under conditions suitable for complex formation The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric, means. Quantities of TBS expressed insubject, control and disease, samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encoding TBSmay be used for diagnostic purposes. The polynucleotides which may beused include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofTBS may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of TBS, andto monitor regulation of TBS levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding TBS or closely related molecules, may be used to identifynucleic acid sequences which encode TBS. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleofides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding TBS, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe TBS encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring TBS.

Means for producing specific hybridization probes for DNAs encoding TBSinclude the cloning of nucleic acid sequences encoding TBS or TBSderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding TBS may be used for the diagnosis ofconditions or disorders which are associated with expression of TBS.

Examples of such conditions or disorders include cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and immune responsedisorders such as AIDS, Addison's disease, adult respiratory distresssyndrome, allergies, anemia, asthma, atherosclerosis, bronchitis,cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, erythema nodosum,atrophic gastritis, glomerulonephritis, gout, Graves' disease,hypereosinophilia, irritable bowel syndrome, lupus erythematosus,multiple sclerosis, myasthenia gravis, myocardial or pericardialinflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis,rheumatoid arthritis, scleroderma, Sjögren's syndrome, and autoimmunethyroiditis; complications of cancer, hemodialysis, and extracorporealcirculation; viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections; and trauma.

The polynucleotide sequences encoding TBS may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dipstick, pIN, ELISA assays or microarraysutilizing fluids or tissues from patient biopsies to detect altered TBSexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding TBS may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingTBS may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding TBS in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of TBS, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes TBS, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding TBS may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced in vitro.Oligomers will preferably consist of two nucleotide sequences, one withsense orientation (5′->3′) and another with antisense (3′<-5′), employedunder optimized conditions for identification of a specific gene orcondition. The same two oligomers, nested sets of oligomers, or even adegenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

Methods which may also be used to quantitate the expression of TBSinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or calorimetricresponse gives rapid quantitation.

In further embodiments, an oligonucleotide derived from any of thepolynucleotide sequences described herein may be used as a target in amicroarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents (Heller, R.et al. (1997) Proc. Natl. Acad. Sci. 94:2150-55).

In one embodiment, the microarray is prepared and used according to themethods described in PCT application WO95/11995 (Chee et al.), Lockhart,D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al.(1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which areincorporated herein in their entirety by reference.

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides which are only 7-10nucleotides in length. The microarray may contain oligonucleotides whichcover the known 5′, or 3′, sequence, sequential oligonucleotides whichcover the full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The“pairs” will be identical, except for one nucleotide which preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/25 1116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray, the RNA or DNAfrom a biological sample is made into hybridization probes. The mRNA isisolated, and cDNA is produced and used as a template to make antisenseRNA (aRNA). The aRNA is amplified in the presence of fluorescentnucleotides, and labeled probes are incubated with the microarray sothat the probe sequences hybridize to complementary oligonucleotides ofthe microarray. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray. The biological samples may be obtained from any bodilyfluids (such as blood, urine, saliva, phlegm, gastric juices, etc.),cultured cells, biopsies, or other tissue preparations. A detectionsystem may be used to measure the absence, presence, and amount ofhybridization for all of the distinct sequences simultaneously. Thisdata may be used for large scale correlation studies on the sequences,mutations, variants, or polymorphisms among samples.

In another embodiment of the invention, the nucleic acid sequences whichencode TBS may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome or to artificial chromosome constructions, such as humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions orsingle chromosome cDNA libraries as reviewed in Price, C. M. (1993)Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

Fluorescent in situ hybridization (FISH as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in various scientific journals or at Online Mendelian Inheritancein Man (OMIM). Correlation between the location of the gene encoding TBSon a physical chromosomal map and a specific disease , or predispositionto a specific disease, may help delimit the region of DNA associatedwith that genetic disease. The nucleotide sequences of the subjectinvention may be used to detect differences in gene sequences betweennormal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, TBS, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between TBSand the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to TBS large numbers of differentsmall test compounds are synthesized on a solid substrate, such asplastic pins or some other surface. The test compounds are reacted withTBS, or fragments thereof, and washed. Bound TBS is then detected bymethods well known in the art. Purified TBS can also be coated directlyonto plates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding TBS specificallycompete with a test compound for binding TBS. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with TBS.

In additional embodiments, the nucleotide sequences which encode TBS maybe used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I KIDNNOT02 cDNA Library Construction

The KIDNNOT02 cDNA library was constructed from a kidney obtained fromthe Keystone Skin Bank, International Institute for the Advancement ofMedicine (Exton, Pa.). Kidney tissue from a 64 year old Caucasian female(Lot HEF698) was flash frozen, ground in a mortar and pestle, and lysedimmediately in buffer containing guanidinium isothiocyanate. Lysis wasfollowed by several phenol chloroform extractions and ethanolprecipitation. Poly A +RNA was isolated using biotinylated oligo d(T)primer and streptavidin coupled to a paramagnetic particle (PromegaCorp, Madison Wis.) and sent to Stratagene.

Stratagene prepared the cDNA library using oligo d(T) priming. Syntheticadapter oligonucleotides were ligated onto the cDNA molecules, whichwere then inserted into the Uni ZAP-vector system (Stratagene). Thequality of the cDNA library was screened using DNA probes, and thepBluescript phagemid (Stratagene) was excised. Subsequently, libraryphage particles were infected into E. coli host strain XL1-Blue(Stratagene). Alternative unidirectional vectors might include, but arenot limited to, pcDNAI (Invitrogen, San Diego, Calif.) and pSH1ox-1(Novagen, Madison Wis.).

II Isolation and Sequencing of cDNA Clones

The phagemid forms of individual cDNA clones were obtained by the invivo excision process, in which the host bacterial strain was coinfectedwith both the lambda library phage and an f1 helper phage. Polypeptidesderived from both the library-containing phage and the helper phagenicked the lambda DNA, initiated new DNA synthesis from definedsequences on the lambda target DNA, and created a smaller, singlestranded circular phagemid DNA molecule containing all DNA sequences ofthe pBluescript® plasmid and the cDNA insert. The phagemid DNA wassecreted from the cells and purified and was, then used to re-infectfresh host cells. Double stranded phagemid DNA was produced and selectedon medium containing ampicillin.

Phagemid DNA was purified using the Magic Minipreps™ DNA PurificationSystem (catalogue #A7100. Promega Corp., Madison, Wis.). Alternatively,phagemid DNA was purified using the QIAwell-8, QIAwell PLUS, and QIAwellULTRA DNA Purification System (QIAGEN). The DNA was eluted from thepurification resin already prepared for DNA sequencing and otheranalytical manipulations.

The cDNAs were sequenced by the method of Sanger F. and A. R. Coulson(1975; J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200(Hamilton, Reno Nev.) in combination with four Peltier Thermal Cyclers(PTC200; MJ Research, Watertown Mass.) and Applied Biosystems 377 or 373DNA Sequencing Systems (Perkin Elmer), and reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences of the Sequence Listing or the amino acidsequences deduced from them were used as query sequences againstdatabases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases, which contain previously identified and annotated sequences,were searched for regions of homology (similarity) using BLAST (BasicLocal Alignment Search Tool) (Altschul S. F. (1993) J. Mol. Evol.36:290-300; Altschul, S. F. et al. (1990) J. Mol. Biol 215:403-10).

BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal or plant) origin. Other algorithms, such asthe one described in Smith R. F. and T. F. Smith (1992 ProteinEngineering 5:35-51), incorporated herein by reference, can be used whendealing with primary sequence patterns and secondary structure gappenalties. As disclosed in this application, the sequences have lengthsof at least 49 nucleotides, and no more than 12% uncalled bases (where Nis recorded rather than A, C, G, or T).

The BLAST approach, as detailed in Karlin and Altschul (1993; Proc.Natl. Acad. Sci. 90:5873-7) and incorporated herein by reference,searches matches between a query sequence and a database sequence, toevaluate the statistical significance of any matches found, and toreport only those matches which satisfy the user-selected threshold ofsignificance. In this application, threshold was set at 10-25 fornucleotides and 10-14 for peptides.

Incyte nucleotide sequence were searched against the GenBank databasesfor primate (pri), rodent (rod), and mammalian (mam) sequences, anddeduced amino acid sequences from the same clones are searched againstGenBank functional protein databases, mammalian (mamp), vertebrate(vrtp) and eukaryote (eukp), for homology.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involving the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. (1993)J.Mol.Evol. 36:290-300; Altschul, S. F. et al. (1990) J.Mol.Evol.215:403-410) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:

% sequence identity×% maximum BLAST score/100

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1-2% error;and at 70, the match will be exact. Homologous molecules are usuallyidentified by selecting those which show product scores between 15 and40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding TBS occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of TBS Encoding Polynucleotides

The nucleic acid sequence of the Incyte Clone 194046 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension in theantisense direction, and the other was synthesized to extend sequence inthe sense direction. Primers were used to facilitate the extension ofthe known sequence “outward” generating amplicons containing new,unknown nucleotide sequence for the region of interest. The initialprimers were designed from the cDNA using OLIGO 4.06 (NationalBiosciences), or another appropriate program, to be about 22 to about 30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures of about 68° to about 72°C. Any stretch of nucleotides which would result in hairpin structuresand primer-primer dimerizations was avoided.

Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. Beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, PCR was performedusing the Peltier Thermal Cycler (PTC200; M.J. Research, Watertown,Mass.) and the following pararneters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat step 4-6 for 15 additional cyclesStep 8 94° C. for 15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15min Step 11 Repeat step 8-10 for 12 cycles Step 12 72° C. for 8 min Step13 4° C. (and holding)

A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQuick™ (QIAGEN Inc., Chatsworth, Calif.), and trimmedof overhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2×Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2×Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for an additional29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (and holding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5′ regulatory sequences using the procedure above, oligonucleotidesdesigned for 5′ extension, and an appropriate genomic library.

VI Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1,or Pvu II; DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots, or the blots areexposed in a PhosphorImager cassette (Molecular Dynamics, Sunnyvale,Calif.) hybridization patterns are compared visually.

VII Microarrays

To produce oligonucleotides for a microarray, the nucleotide sequencedescribed herein is examined using a computer algorithm which starts atthe 3′ end of the nucleotide sequence. The algorithm identifiesoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that would interfere with hybridization. Thealgorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein byreference). In another alternative, a “gridded” array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

VIII Complementary Polynucleotides

Sequence complementary to the TBS-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring TBS. Although use of oligonucleotides comprising from about 15to about 30 base-pairs is described, essentially the same procedure isused with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of TBS, SEQ ID NO:1. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the TBS-encoding transcript.

IX Expression of TBS

Expression of TBS is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express TBS in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion of TBSinto the bacterial growth media which can be used directly in thefollowing assay for activity.

X Demonstration of TBS Activity

TBS can be expressed in a mammalian cell line such as 293T bytransfecting with an eukaryotic expression vector encoding TBS.Eukaryotic expression vectors are commercially available, and thetechniques to introduce them into cells are well known to those skilledin the art. A small amount of a second plasmid, which expresses any oneof a number of reporter genes such as β-galactosidase, is co-transformedinto the cells in order to allow rapid identification of those cellswhich have taken up and expressed the foreign DNA. The cells arecultured in a defined synthetic medium with concentrations of GTP for atleast 48 hours after transformation to allow expression and accumulationof TBS and β-galactosidase.

Transformed cells expressing β-galactosidase are stained blue when asuitable colorimetric substrate is added to the culture media underconditions that are well known in the art. Increasing concentrations ofGTP induces increasing numbers of reporter gene positive cells (Ren, M.et al. (1996) Proc. Natl. Acad. Sci. 93: 5151-5155). GTP-treated cellswhich were not transformed with the TBS expression vector are used ascontrols, as are TBS transfected cells cultured without supplementalGTP.

XI Production of TBS Specific Antibodies

TBS that is substantially purified using PAGE electrophoresis (Sambrook,supra), or other purification techniques, is used to immunize rabbitsand to produce antibodies using standard protocols. The amino acidsequence deduced from SEQ ID NO:2 is analyzed using DNASTAR software(DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regions,is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio iodinated, goat anti-rabbitIgG.

XII Purification of Naturally Occurring TBS Using Specific Antibodies

Naturally occurring or recombinant TBS is substantially purified byimmunoaffinity chromatography using antibodies specific for TBS. Animmunoaffinity column is constructed by covalently coupling TBS antibodyto an activated chromatographic resin, such as CNBr-activated Sepharose(Pharmacia & Upjohn). After the coupling, the resin is blocked andwashed according to the manufacturer's instructions.

Media containing TBS is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof TBS (e.g., high ionic strength buffers in the presence of detergent).The column is eluted under conditions that disrupt antibody/TBS binding(e.g., a buffer of pH 2-3 or a high concentration of a chaotrope, suchas urea or thiocyanate ion), and TBS is collected.

XIII Identification of Molecules Which Interact with TBS

TBS or biologically active fragments thereof are labeled with ¹²⁵IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled TBS, washed and any wells withlabeled TBS complex are assayed. Data obtained using differentconcentrations of TBS are used to calculate values for the number,affinity, and association of TBS with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

3 305 amino acids amino acid single linear KIDNNOT02 194046 1 Met ThrAsn Gln Tyr Gly Ile Leu Phe Lys Gln Glu Gln Ala His Asp 1 5 10 15 AspAla Ile Trp Ser Val Ala Trp Gly Thr Asn Lys Lys Glu Asn Ser 20 25 30 GluThr Val Val Thr Gly Ser Leu Asp Asp Leu Val Lys Val Trp Lys 35 40 45 TrpArg Asp Glu Arg Leu Asp Leu Gln Trp Ser Leu Glu Gly His Gln 50 55 60 LeuGly Val Val Ser Val Asp Ile Ser His Thr Leu Pro Ile Ala Ala 65 70 75 80Ser Ser Ser Leu Asp Ala His Ile Arg Leu Trp Asp Leu Glu Asn Gly 85 90 95Lys Gln Ile Lys Ser Ile Asp Ala Gly Pro Val Asp Ala Trp Thr Leu 100 105110 Ala Phe Ser Pro Asp Ser Gln Tyr Leu Ala Thr Gly Thr His Val Gly 115120 125 Lys Val Asn Ile Phe Gly Val Glu Ser Gly Lys Lys Glu Tyr Ser Leu130 135 140 Asp Thr Arg Gly Lys Phe Ile Leu Ser Ile Ala Tyr Ser Pro AspGly 145 150 155 160 Lys Tyr Leu Ala Ser Gly Ala Ile Asp Gly Ile Ile AsnIle Phe Asp 165 170 175 Ile Ala Thr Gly Lys Leu Leu His Thr Leu Glu GlyHis Ala Met Pro 180 185 190 Ile Arg Ser Leu Thr Phe Ser Pro Asp Ser GlnLeu Leu Val Thr Ala 195 200 205 Ser Asp Asp Gly Tyr Ile Lys Ile Tyr AspVal Gln His Ala Asn Leu 210 215 220 Ala Gly Thr Leu Ser Gly His Ala SerTrp Val Leu Asn Val Ala Phe 225 230 235 240 Cys Pro Asp Asp Thr His PheVal Ser Ser Ser Ser Asp Lys Ser Val 245 250 255 Lys Val Trp Asp Val GlyThr Arg Thr Cys Val His Thr Phe Phe Asp 260 265 270 His Gln Asp Gln ValTrp Gly Val Lys Tyr Asn Gly Asn Gly Ser Lys 275 280 285 Ile Val Ser ValGly Asp Asp Gln Glu Ile His Ile Tyr Asp Cys Pro 290 295 300 Ile 305 1221base pairs nucleic acid single linear KIDNNOT02 194046 2 CGCGGACCCGGCTTCCGACG TGCAGCCTGG CAGTGCAGTG AGCTGTCTGG CCTTTTGTCC 60 TTGATCCTTGGTTAAGGAAA TGACCAACCA GTACGGTATT CTCTTCAAAC AAGAGCAAGC 120 CCATGATGATGCCATTTGGT CAGTTGCTTG GGGGACAAAC AAGAAGGAAA ACTCTGAGAC 180 AGTGGTCACAGGCTCCCTAG ATGACCTGGT GAAGGTCTGG AAATGGCGTG ATGAGAGGCT 240 GGACCTACAGTGGAGTCTGG AGGGACATCA GCTGGGAGTG GTGTCTGTGG ACATCAGCCA 300 CACCCTGCCCATTGCTGCAT CCAGCTCTCT TGATGCTCAT ATTCGTCTTT GGGACTTGGA 360 AAATGGCAAACAGATAAAGT CCATAGATGC AGGACCTGTG GATGCCTGGA CTTTGGCCTT 420 TTCTCCTGATTCCCAGTATC TGGCCACAGG AACTCATGTC GGGAAAGTGA ACATTTTTGG 480 TGTGGAAAGTGGGAAAAAGG AATATTCTTT GGACACGAGA GGAAAATTCA TTCTTAGTAT 540 TGCATATAGTCCTGATGGGA AATACCTAGC CAGTGGAGCC ATAGATGGAA TCATCAATAT 600 TTTTGATATTGCAACTGGAA AACTTCTGCA TACCCTGGAA GGCCATGCCA TGCCCATTCG 660 CTCCTTGACCTTTTCCCCGG ACTCCCAGCT CCTTGTCACT GCTTCAGATG ATGGCTACAT 720 CAAGATCTATGATGTACAAC ATGCCAATTT GGCTGGCACG CTGAGCGGCC ATGCCTCCTG 780 GGTGCTGAACGTTGCATTCT GTCCTGATGA CACTCACTTT GTTTCCAGTT CGTCTGACAA 840 AAGTGTAAAAGTTTGGGATG TTGGAACGAG GACTTGTGTT CACACCTTCT TTGATCACCA 900 GGATCAGGTCTGGGGAGTAA AATACAATGG AAATGGTTCA AAAATTGTGT CTGTTGGAGA 960 TGACCAGGAAATTCACATCT ATGATTGTCC AATTTAAACA TCAAAGTCTC CAGGCTTATG 1020 CTGCAAAGAGAATGTACGGA TTGATCATGA CATTCCTTAC CTTCTTAGGC TTGTTTAAAA 1080 GAAATATAGCATTTATTGTA GCAAAGACTT AAATTTTGTA GATACAATAT GAATCTTTTC 1140 ATGTTTTATTGGAAATGCTG TTCATACTTT AACATAAAGC TTTCTTAATG CAAAAAGAAA 1200 AAAAAAAAAAAAAAAAAAGA G 1221 302 amino acids amino acid single linear GenBank1079671 3 Met Arg Lys Glu Tyr Leu Val Ser His Ile Glu Glu Asn Ala HisGln 1 5 10 15 Ala Asp Ile Tyr Ser Leu Asn Val Val Ala Gly Asn Leu TrpSer Ala 20 25 30 Ser Gly Asp Ser Lys Ile Lys Lys Trp Ser Ile Gly Asp AlaGlu His 35 40 45 Ser Leu Val Glu Glu Ile Asp Thr Pro His Lys Leu Gly ValHis His 50 55 60 Leu Ala Thr Ser Leu Asp Glu Asn Val Val Val Ser Cys GlyPhe Gly 65 70 75 80 Gln Asp Val Tyr Val Trp Asn Pro Glu Thr Asn Glu PheArg Asp Leu 85 90 95 Gly Asn Asn Ala Gln His Pro Ser Glu Cys Trp Ser SerCys Ile Ser 100 105 110 Pro Asp Gly Gln Thr Ile Ala Phe Thr Ser Val AspGly Arg Ile Ala 115 120 125 Val Trp Asp Asn Pro Ser Asp Cys Lys Ile SerGlu Leu Asp Thr Lys 130 135 140 Gly Lys Phe Gly Leu Cys Ile Asp Tyr SerPro Asn Gly Arg Phe Ile 145 150 155 160 Val Ser Gly His Gln Thr Gly GlnLeu Phe Leu Ile Ser Thr Glu Thr 165 170 175 Gly Arg Leu Phe His Val LeuSer Gly His Thr Ser Pro Val Arg Ser 180 185 190 Val Ala Phe Ser Pro GlySer Thr Leu Leu Ala Ala Ala Gly Asp Ser 195 200 205 Lys Met Ile Thr IleTyr Asp Val Leu Ser Gly Asp Gln Val Gly Gln 210 215 220 Leu Arg Gly HisAla Ala Trp Ile Phe Ala Val Ala Phe Asn Pro Val 225 230 235 240 Gly AspLeu Leu Leu Ser Ala Asp Val Glu Gly Lys Ile Lys Ile Trp 245 250 255 AspIle Asp Thr Met Glu Cys Ile Ser Thr Gln Ser Glu Thr Asp Gly 260 265 270Ala Ile Trp Ala Val Ala Trp Tyr Lys Asn Gly Phe Ile Val Ala Gly 275 280285 Ala Asp Lys Ser Ile Arg Trp Tyr Arg Ala Ala Ala Thr Glu 290 295 300

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: a) the amino acidsequence as shown in SEQ ID NO:1, and b) an immunogenic fragment of theamino acid sequence as shown in SEQ ID NO:1, wherein said fragmentcomprises at least 15 contiguous amino acids of the amino acid sequenceas shown in SEQ ID NO:1, that can produce an antibody specific to SEQ IDNO:1.
 2. An isolated polypeptide of claim 1 comprising the amino acidsequence as shown in SEQ ID NO:1.
 3. A composition comprising apolypeptide of claim 1 and a pharmaceutically acceptable excipient.
 4. Acomposition of claim 3, wherein the polypeptide comprises the amino acidsequence as shown in SEQ ID NO:1.
 5. A method for screening a compoundfor effectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 6. A methodfor screening a compound for effectiveness as an antagonist of apolypeptide of claim 1, the method comprising: a) exposing a samplecomprising a polypeptide of claim 1 to a compound, and b) detectingantagonist activity in the sample.
 7. A method of screening for acompound that specifically binds to the polypeptide of claim 1, saidmethod comprising the steps of: a) combining the polypeptide of claim 1with at least one test compound under suitable conditions, and b)detecting binding of the polypeptide of claim 1 to the test compound,thereby identifying a compound that specifically binds to thepolypeptide of claim
 1. 8. A method of screening for a compound thatmodulates the activity of the polypeptide of claim 1, said methodcomprising: a) combining the polypeptide of claim 1 with at least onetest compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim 1.